PROJECT SUMMARY Nasal carriers of Staphylococcus aureus can serve as reservoirs for this opportunistic pathogen and are them- selves at increased risk for invasive infection. Our long-term goal is to develop probiotic-based strategies to block colonization by antibiotic-resistant opportunistic pathogens. It is the objective of this project to identify nasal commensal bacteria associated with sustained S. aureus exclusion. Our central hypothesis is that nasal commensals?such as Dolosigranulum and other yet-to-be-identified bacteria?can effectively and stably ex- clude S. aureus nasal carriage. The rationale for this project is that recent cross-sectional studies support the potential for using probiotics to reduce S. aureus carriage; but to progress to the next step, it is crucial for us to elucidate the intra- and interspecies dynamics of nasal microbiota and to determine if and which commensals can stably exclude S. aureus. This research is made possible by our team?s unique combination of expertise in microbiome and genomic research, complex ecological data analysis, and novel nasal microbiome-epithelium culture models, as well our team?s unique access to population-based nasal sampling. We will leverage these resources to accomplish the following specific aims: Aim 1. Identify nasal commensals associated with stable exclusion of S. aureus in healthy adults. We will enroll 1,000 healthy community-dwelling adults and follow 180 individuals (with equal represen- tation of each nasal community) over a 12-month period to evaluate the dynamics of nasal bacterial ecology using culture-based methods, real-time PCR, genomics, and ecological analyses. Aim 2. Identify nasal commensals associated with sustained MRSA decolonization after mupi- rocin treatment. We will enroll and follow 100 MRSA carriers undergoing decolonization and 50 persistent S. aureus nasal carriers from Aim 1 as controls for 12-months to determine the pre- and post-treatment nasal communities associated with decolonization success using culture-based methods, real-time PCR, genomics, and ecological analyses. Aim 3. Demonstrate exclusion of S. aureus by specific nasal communities and individual nasal commensals in vitro. Using samples and nasal bacterial isolates from Aims 1 and 2, we will reconstitute the major nasal communities in a novel microbiome-nasal epithelium culture model and verify if some nasal com- mensals can resist or exclude S. aureus. The approach is innovative, in this team?s opinion; because it will examine the nasal microbiome dynamics from the perspective of absolute abundance and it will verify observational results from two large cohort studies in a novel in vitro system. The project is significant because it has the potential to build a new nasal decoloni- zation strategy that can stably exclude S. aureus without selecting for antibiotic resistance. The study will gen- erate genomic data, model systems, and isolate collections with exceptional translational potential.